Component of bromelain

ABSTRACT

The invention relates to a component of bromelain which is largely responsible for the ability of bromelain to interrupt the MAP kinase cascade. The component contains ananain and comosain and is useful in the treatment or prevention of diseases and conditions mediated by T cell activation or by activation of the MAP kinase pathway.

[0001] The present invention relates to a component of bromelain. Inparticular, the invention relates to the use of this bromelain componentin medicine, particularly as an anti-cancer agent and animmunosuppressive agent.

[0002] Stem bromelain (bromelain) is the collective name for theproteolytic enzymes found in the tissues of the plant Bromeliaceae. Itis a mixture of various moieties derived from the stem of the pineappleplant (Ananas comosus). Bromelain is known to contain at least fiveproteolytic enzymes but also non-proteolytic enzymes, including an acidphosphatase and a peroxidase; it may also contain amylase and cellulaseactivity. In addition, various other components are present.

[0003] Bromelain has previously been used in the treatment of a varietyof conditions including inflammation and, in particular, it has beenused in the treatment of diarrhoea. The use of bromelain in thetreatment of infectious diarrhoea is described in WO-A-9301800, where itis suggested that bromelain works by destroying intestinal receptors forpathogens by proteolysis, and in WO-A-8801506, which teaches thatbromelain detaches pathogens from intestinal receptors.

[0004] Taussig et al, Planta Medica, 1985, 538-539 and Maurer et al,Planta Medica, 1988, 377-381 both suggest that bromelain may be of usein inhibiting tumour growth. U.S. Pat. No. 5,223,406, DE-A-4302060 andJP-A-59225122 also teach the use of bromelain in the treatment ofcancer. U.S. Pat. No. 5,223,406 teaches that bromelain is capable ofinducing tumour necrosis factor (TNF) while DE-A-4302060 teaches thatbromelain can prevent metastasis by the structural modification of thetumour surface protein CD44.

[0005] In WO-A-9400147, various experiments were described whichdemonstrate that proteolytic enzymes and, in particular, bromelain, arecapable of inhibiting secretion. The application also discloses thatbromelain can reduce toxin binding activity and can inhibit thesecretory effect of toxins such as heat labile toxin (LT) and choleratoxin (CT) and also toxins such as heat stable toxin (ST). This is inspite of the fact that ST has a very different mode of action from LTand CT. These observations were explained by the fact that one componentof the bromelain mixture, stem bromelain protease, appears to be capableof modulating cyclic nucleotide pathways and this is discussed furtherin WO-A-9500169. In addition. bromelain has also been demonstrated toinhibit secretion caused by the calcium dependent pathway.

[0006] The present inventors have studied the varied biological effectsof bromelain and, in particular, its effects in a well documented modelof intracellular signal transduction, namely T cell receptor (TCR)/CD3signalling and IL-2 production. Significant progress over recent yearshas led to the understanding of biochemical events which occur followingTCR engagement (reviewed Cantrell, Annu. Rev. Immunol. 14, 259-274,(1996)), therefore TCR signalling provides an excellent model forelucidation of the effects of biologically active compounds. Effective Tcell activation requires two signals. The first signal is generated bythe TCR/CD3 complex after engagement with antigen peptide presented bythe major histocompatibility complex (MHC) expressed on antigenpresenting cells (APC) (Cantrell, 1996). The second, costimulatorysignal is generated by ligation of CD28 receptors on T cells with the B7family of ligands on APC. A key element in the signalling pathwayinvolved in transducing receptor-initiated signals to the nucleus is thefamily of mitogen-activated protein kinases (MAPk). The best studied ofthese kinases are the extracellular signal-regulated protein kinases(ERK)-1 and ERK-2 (also referred to as p44^(MAPk) and p42^(MAPk),respectively). ERK's are serine/threonine kinases that are activatedwhen phosphorylated on tyrosine and threonine residues. In vitro, thisactivation is reversed if either residue is dephosphorylated. Arelatively newly discovered member of the MAPk family are c-JunNH₂-terminal kinases (JNKs) which exist as 46 kDa and 55 kDa forms thatalso require phosphorylation for activation. ERK activation is dependenton p56^(Lck) and coupling of the TCR/CD3 complex to p21^(Ras), withsubsequent activation of the Raf-1/MEK1/ERK kinase cascade. JNKactivation also requires p21^(Ras), as well as signals generated by theCD28 costimulatory receptor which activate GTP (guanosinetriphosphate)-binding proteins (such as Rac1 or Cdc42) that induce thePAK/MEKK/SEK/JNK kinase cascade. Activated ERK phosphorylates Elk-1,which in turn, mediates induction of c-fos activity followingphosphorylation of c-jun by JNK. Activated c-fos and c-jun combine toform the AP-1 protein required for IL-2 synthesis. The above events aresummarised in FIG. 1. All the above-mentioned signalling events requiretyrosine phosphorylation, as inhibitors of protein tyrosine kinases(PTKs) inhibit many events associated with TCR stimulation, including Tcell activation and IL-2 production.

[0007] In WO-A-9600082, we showed that bromelain could inhibit tyrosinephosphorylation and activation of ERK-2 in T cells stimulated via theTCR, or with combined phorbol ester plus calcium ionophore. We have nowfound that, in association with decreased ERK activity, bromelaindecreased IL-2, IL-4 and IFN-γ mRNA accumulation in T cells stimulatedwith phorbol ester and ionophore, but did not affect cytokine mRNAaccumulation in cells stimulated via the TCR. This data suggests theexistence of a TCR-activated, ERK-independent pathway involved incytokine production in T cells.

[0008] From the prior art, it is clear that bromelain is a mixture whichhas a variety of different physiological effects. Not all of thecomponents of the bromelain mixture have been characterised and so,except for stem bromelain protease, whose activity we have described, itis not clear which of the components is responsible for which of thevarious different effects of bromelain. This is, of course, a majordisadvantage if the bromelain mixture is to be administered as apharmaceutical because while one component of bromelain might give thedesired effect, there may well be unwanted side effects arising from theaction of some other component of the bromelain mixture.

[0009] It would therefore be beneficial if individual components ofbromelain giving rise to particular medicinal activities could beisolated and administered separately so as to lessen the possibility ofside effects. We have now identified an active fraction of crudebromelain which is responsible for its ability to inhibit ERKactivation, and therefore block the MAP kinase pathway. Although not asingle protein, this fraction consists of only a few components and sothe possibility of side effects when it is administered to patients isgreatly reduced compared with crude bromelain.

[0010] The fraction of the invention, which the inventors havedesignated CCS, may be isolated from the bromelain mixture byconventional methods, for example by chromatography. High performanceliquid chromatograpy (HPLC) is suitable for the purpose and particularlygood separation of bromelain proteins may be achieved by fast proteinliquid chromatography (FPLC™) using a column packing material such asS-sepharose. As will be described in more detail in the examples, inchromatography on S-sepharose using a linear gradient of 0 to 0.8Msodium chloride in acetate buffer over 300 ml, the protein of thepresent invention was the last double peak off the column.

[0011] In a first aspect of the present invention, there is provided acomponent of bromelain which contains proteins having molecular weightsof about 15.07 kDa, 25.85 kDa and 27.45 kDa as determined by SDS-PAGE,has isoelectric points of 10.4 and 10.45 and is obtainable by thefollowing method:

[0012] i. dissolving bromelain in acetate buffer at pH 5.0;

[0013] ii. separating the components of the bromelain by fast flow highperformance liquid chromatography on S-sepharose eluting with a lineargradient of 0 to 0.8 M sodium chloride in acetate buffer over 300 ml;

[0014] iii. collecting the fraction corresponding to the final doublepeak off the column; and

[0015] iv. isolating the protein from the fraction collected in (iii).

[0016] This fraction, termed CCS, has been found to have a number ofpotentially useful activities. Firstly, we have found that it blocksERK-2 phosphorylation, and therefore the MAP kinase cascade. Inaddition, it blocked IL-2 production and CD4⁺ T cell proliferation.However, CCS did not affect splenocyte proliferation, which suggeststhat it has a selective mode of action. CCS also differentially blockedgrowth of human tumour cell lines including ovarian, lung, colon,melanoma and breast tumours. The differential activity of CCS againstthe different cell lines further suggests that CCS has a selective modeof action and that it may also act as an anti-cancer agent. Theinhibitory effect of CCS on ERK-2 is dependent on its proteolyticactivity, since E-64, a selective cysteine protease inhibitor, couldabrogate the effect of CCS.

[0017] Although in WO-A-9724138, we stated that proteases in general arecapable of decreasing MAP kinase activation, we have now found that thisis not the case as trypsin does not abrogate T cell signalling and,indeed, in other studies, has been shown to increase MAPk activation(Belham et al, 1996, Biochem. J., 320, 939-946). Thrombin, a proteaseinvolved in the blood coagulation cascade, has also been shown toincrease MAP kinase activation (Vouret-Craviari et al, 1993, Biochem.J., 289, 209-214). The inventors have now also shown that otherproteases contained within the crude bromelain mixture do not block theactivation of the MAP kinase pathway.

[0018] It is possible that the effects of CCS on the MAP kinase pathwayin T cells are mediated by specific proteolytic effects at the cellsurface. It is known that bromelain cleaves the CD45 RA isoform andselectively removes other surface molecules from human PBMCs. Bromelainalso partially removes CD4 from T cell surfaces. Since CD45 and CD4 playan obligate stimulatory role in TCR-mediated T cell activation, CCS mayinterfere with TCR signalling by affecting these molecules. Although theimportance of CD45 and CD4 is well recognised for TCR-initiated signaltransduction, it is possible to bypass their requirements for T cellactivation by the use of phorbol ester and calcium ionophore. Use ofcombined phorbol esters and ionophore restores normal function to Tcells which have been made refractory to TCR stimulation by the use oftyrosine kinase inhibitors or which are CD45 or p56^(Lck) deficient.

[0019] However, in the present study, the inventors have shown thatnormal function is not restored to T cells pre-treated with CCS whenthey are treated with PMA plus ionophore. The inhibitory effect of CCSon ERK-2 is thus not thought to be mediated via effects on CD45 or CD4on T cells. CCS possibly affects an as yet unidentified surfacemolecule, which, in turn, affects the MAP kinase pathway. The inhibitoryeffect of CCS on cytokine production is thus not thought to be mediatedvia its effects on CD45 or CD4 on T cells. The inhibitory effect of CCSon T cell signal transduction was not because of toxicity of thecompound, since CCS did not affect splenocyte or GA15 cell viability.The viability of the cells was not significantly affected by culture inthe presence of CCS for periods of time greater than 48 hours.

[0020] Since we have shown that fraction CCS from crude bromelain blocksactivation of the MAP kinase pathway and blocks T cell activation, CCSmay be of use in the treatment of T cell-mediated diseases.

[0021] In addition to its importance for IL-2 production and T cellactivation, the MAP kinase pathway is also important for the productionof growth factors such as epidermal growth factor (EGF), plateletderived growth factor (PGDF) and insulin-like growth factor (IGF). CCSwill therefore block the production of these, and other, growth factorsand the production of other cytokines such as IL-4, IFN-γ, GM-GSF andmany more.

[0022] Also, as briefly mentioned above, CCS is likely to be of use inthe treatment of cancer.

[0023] Thus, CCS may be of use in a method for the treatment orprevention of diseases or conditions mediated by:

[0024] i. activation of T cells;

[0025] ii. activation of the MAP kinase pathway; or

[0026] iii. the production of growth factors or cytokines;

[0027] or in the treatment or prevention of cancer.

[0028] In a second aspect of the invention, therefore, there is provideda component of bromelain which contains proteins having molecularweights of about 15.07 kDa, 25.85 kDa and 27.45 kDa as determined bySDS-PAGE, has isoelectric points of 10.4 and 10.45 and is obtainable bythe following method:

[0029] i. dissolving bromelain in acetate buffer at pH 5.0;

[0030] ii. separating the components of the bromelain by fast flow highperformance liquid chromatography on S-sepharose eluting with a lineargradient of 0 to 0.8 M sodium chloride in acetate buffer over 300 ml;

[0031] iii. collecting the fraction corresponding to the final doublepeak off the column; and

[0032] iv. isolating the protein from the fraction collected in (iii)

[0033] for use in medicine, particularly in the treatment or preventionof diseases and conditions mediated by:

[0034] i. activation of T cells;

[0035] ii. activation of the MAP kinase pathway; or

[0036] iii. the production of growth factors or cytokines;

[0037] or in the treatment or prevention of cancer.

[0038] On further analysis of the CCS fraction of bromelain, the presentinventors have found that it comprises more than one component.Sequencing of the proteins in the fraction showed that it consists ofthe cysteine proteases ananain and comosain together with various othercomponents.

[0039] Thus, it appears that both ananain and comosain or a mixture ofthe two may be responsible for the activity of the CCS fraction ofbromelain.

[0040] In a further aspect of the invention, therefore, there isprovided the use of ananain, comosain, a mixture of ananain and comosainor the CCS fraction of bromelain in the preparation of an agent fortreatment or prevention of diseases or conditions mediated by:

[0041] i. activation of T cells;

[0042] ii. activation of the MAP kinase pathway; or

[0043] iii. the production of growth factors or cytokines;

[0044] or in the treatment or prevention of cancer.

[0045] In our earlier application WO-A-9600082 we discussed theinhibition of the MAP kinase cascade by crude bromelain. However, atthat time, we were not able to determine which component of the crudebromelain mixture was responsible for this activity although wespeculated that it might be stem bromelain protease. We have nowdiscovered that in addition to blocking cyclic nucleotide pathways, stembromelain protease does have some activity against the MAP kinasepathway. However, it is far less effective in blocking the MAP kinasecascade than the CCS fraction of bromelain of the present invention.Indeed, we have now found that the CCS fraction of bromelain is in theregion of ten orders of magnitude more active than stem bromelainprotease in blocking MAP kinase activation.

[0046] The activation of the MAP kinase pathway in T cells to produceIL-2 and drive T cell clonal expansion is an essential component of theimmune response. The absence of this process can have fatalconsequences, as can be observed in people with AIDS or geneticmutations which result in T cell defects. However, the activation of Tcells can also lead to detrimental consequences. For example, ifautoreactive T cells are activated, autoimmune diseases can result. CCSis therefore likely to be of use in the treatment of autoimmune diseasessuch as rheumatoid arthritis, type-1 diabetes mellitus, multiplesclerosis, Crohn's disease and lupus.

[0047] Also, the activation of T cells specific for engrafted tissue canlead to graft or transplant rejection and so CCS may also be of use inpreventing this.

[0048] The activation of allergen-specific T cells can cause allergicreactions. Inflammatory cytokines and other cellular products, such ashistamine, are released from cells following exposure to allergens. Therelease of histamine and inflammatory cytokines involves the MAP kinasepathway and so blocking of the MAP kinase pathway with CCS is likely tobe an effective treatment for allergies.

[0049] In addition, CCS is likely to be of use in the prevention oftoxic shock and other diseases mediated by over production of bacterialendotoxins. Toxic shock is mediated by the production oflipopolysaccharides (LPS) from gram-negative bacteria. LPS triggers theproduction of TNF-α and interleukin-1 via activation of the MAP kinasepathway in macrophages. The secretion of these cytokines elicits acascade of cytokine production from other cells of the immune system(including T cells), which leads to leucocytosis, shock, intravascularcoagulation and death.

[0050] A further use for CCS is in the prevention of programmed celldeath (apoptosis). This is a special event whereby cells are stimulatedto destroy their own DNA and die. It is an essential event in mostimmune responses (to prevent the accumulation of too many cells), butcan also have immunosuppressive consequences in some instances, such asin HIV infection and ageing so that too many cells die and there areinsufficient left to combat infection (Perandones et al, 1993, J.Immunol., 151, 3521-3529). Because the initiation of apoptosis isdependent on specific cell signalling events, including activation ofthe MAP kinase pathway, CCS is likely to be effective in blockingapoptosis.

[0051] The continual activation of T cells during chronic disease canalso lead to pathological consequences, as can be found in certainchronic parasitic infections, such as chronic granulatomas diseases suchas tuberculoid leprosy, schistosomiasis and visceral leishmaniasis.Furthermore, the invasion of parasites and pathogens, and theirsubsequent survival in cells, is dependent on these organisms utilisinghost cell signalling pathways (Bliska et al, 1993, Cell, 73, 903-920).For example, Salmonella has been demonstrated to phosphorylate MAPkinase, which allows for the bacteria to become endocytosed bymacrophages (Galan et al, 1992, Nature, 357, 588-589). The bacteria thenproliferate and destroy the cell. Because CCS has been shown to modifyhost signalling pathways, and, in particular, to inhibit MAP kinase,another of its potential applications could be to inhibit invasion byparasites and pathogens and their survival in cells.

[0052] CCS may also be of use for the treatment of cancer and, indeed,we have shown that CCS can block human tumour growth in vitro. Theanti-tumour mechanism of action of CCS remains to be determined butseems likely to be a result of the blocking of activation of the ERK-2pathway.

[0053] As mentioned earlier, MAP kinase activation is dependent onp21^(Ras) and Raf-1, which are important oncogenes. p21^(Ras) and Raf-1proteins help to relay signals from growth factor receptors on thesurface of cells to MAP kinases to stimulate cell proliferation ordifferentiation. Oncogenic (or mutant) p21^(Ras) or Raf-1 genes producedefective proteins that have acquired independence from externallysupplied growth factors and, at the same time, may no longer respond toexternal growth-inhibitory signals. Mutant p21^(Ras) or Raf-1 proteinsare thus persistently hyperactive and their unbridled catalytic activityhas a detrimental effect on the control of cell growth. Oncogenicp21^(Ras) or Raf-1 genes therefore promote cancer and tumour formationby disrupting the normal controls on cell proliferation anddifferentiation. Approximately 30% of human cancers have mutations in ap21^(Ras) gene.

[0054] Given that signals transmitted by p21^(Ras) and Raf-1 can beblocked via MAP kinase, CCS would be expected to block cancer and tumourgrowth. The protein fraction of the present invention would therefore beuseful for treating many different types of cancer including solidcancers such as ovarian, colon, breast or lung cancer and melanoma aswell as non-solid tumours and leukaemia.

[0055] The bromelain fraction of the invention will usually beformulated before administration to patients and so, in a further aspectof the invention there is provided a pharmaceutical or veterinarycomposition comprising the CCS fraction of bromelain together with apharmaceutically or veterinarily acceptable excipient.

[0056] The CCS fraction may be administered by a variety of routesincluding enteral, for example oral, nasal, buccal, topical or analadministration or parenteral administration, for example by theintravenous, subcutaneous, intramuscular or intraperitoneal routes.

[0057] In many cases, the oral route may be preferred as this is oftenthe route which patients find most acceptable. The oral route may beparticularly useful if many doses of the protein are required.

[0058] When oral administration is chosen, it may be desirable toformulate the CCS fraction in an enteric-coated preparation in order toassist its survival through the stomach. Alternatively, another orallyadministrable dosage form may be used, for example a syrup, elixir or ahard or soft gelatin capsule, either of which may be enteric coated.

[0059] However, under certain circumstances, it may more convenient touse a parenteral route. For parenteral administration, the protein maybe formulated in distilled water or another pharmaceutically acceptablesolvent or suspending agent.

[0060] A suitable dose of the CCS fraction to be administered to apatient may be determined by the clinician. However, as a guide, asuitable dose may be from about 0.5 to 20 mg per kg of body weight. Itis expected that in most cases, the dose will be from about 1 to 15 mgper kg of body weight and preferably from 1 to 10 mg per kg of bodyweight. For a man having a weight of about 70 kg, a typical dose wouldtherefore be from about 70 to 700 mg.

[0061] The invention will now be further described with reference to thefollowing examples and to the drawings in which:

[0062]FIG. 1 is a diagrammatic representation of signal transductionevents associated with T cell activation that lead to IL-2 production.

[0063]FIG. 2 is an ultra violet elution profile of crude bromelain aftercation exchange chromatography on SP Sepharose high performance media.

[0064]FIG. 3 is a plot showing the proteolytic activity and the proteincontent of crude bromelain fractions after cation exchangechromatography on SP Sepharose high performance media.

[0065]FIG. 4 is an SDS-PAGE of SP Sepharose high performancechromatography pooled fractions run on 4-20% T gradient gels with lanes1 to 4 and 6 to 9 containing proteins CCT, CCV, CCX and CCZ and CCY,CCW, CCU and CCS respectively and lanes 5 and 10 containing molecularweight markers.

[0066]FIG. 5 shows isoelectric focussing of pooled fractions run on pH3-11 gradient gels with Lanes 1, 11 and 12 showing high IEF markers,Lanes 2 and 13 showing crude bromelain and Lanes 3 to 10 showingproteins CCT, CCV, CCX, CCZ, CCY, CCW, CCU and CCS respectively.

[0067]FIG. 6 is a Western blot using anti-phosphotyrosine mAb whichdemonstrates that CCS reduces tyrosine phosphorylation of p42 kDa(ERK-2) protein. Th0 cells were treated with bromelain fractions (50μg/ml) for 30 min, washed and then stimulated with combined PMA (20ng/ml) and ionophore (1μM) for 5 min. Unstimulated cells served ascontrols. Cells were then lysed and postnuclear supernatants weresubjected to SDS-PAGE and Western blotting. In this figure, closedsymbols indicate proteins phosphorylated by combined PMA plus ionophore.Open symbols indicate ERK-2 protein reduced by CCS treatment.

[0068]FIG. 7 is a Western blot using anti-phosphotyrosine mAb whichdemonstrates that CCS increases tyrosine phosphorylation of proteinsubstrates. Th0 cells were treated with CCS, crude bromelain (Brom),stem bromelain protease (SBP) or CCT fraction (50 μg/ml) for 30 min,washed and then stimulated with combined PMA (20 ng/ml) and ionophore (1μM) for 5 min. Unstimulated cells served as controls (Cont). Cells werethen lysed and postnuclear supernatants were subjected to SDS-PAGE andWestern blotting. In this figure, closed symbols indicate proteinsphosphorylated by CCS but not by other treatments. Open symbols indicatephosphoproteins protein reduced by CCS and crude bromelain treatment.

[0069]FIG. 8 is a Western blot using anti-phosphotyrosine mAb whichshows that the inhibitory effect of CCS on ERK- 2 is dependent on itsproteolytic activity and occurs in a dose-dependent manner. Th0 cellswere treated with CCS (0 to 25 μg/ml) or CCS incubated with the selectedprotease inhibitor, E-64, for 30 min. Cells were then washed and thenstimulated with combined PMA (20 ng/ml) and ionophore (1 M) for 5 min.Cells were then lysed and postnuclear supernatants were subjected toSDS-PAGE and Western blotting. In this figure, closed symbols indicateproteins phosphorylated by CCS. Open symbols indicate ERK-2phosphoprotein inhibited by active CCS but not by inactivated CCS.

[0070]FIG. 9 is an immunoblot which confirms that the 42 kDaphosphoprotein inhibited by CCS is ERK-2. Th0 cells were treated withCCS (50 μg/ml) for 30 min, or untreated, washed and then stimulated withcombined PMA (20 ng/ml) and ionophore (1 μM) for 5 min. Cell lysateswere immunoblotted with anti-ERK-2 mAB

[0071]FIG. 10 is a Western blot using anti-phosphotyrosine mAb whichshows that crosslinked anti-CD3E mAb induces tyrosine phosphorylation ofmultiple proteins. Th0 cells were stimulated with crosslinked anti-CD3εfor 0 to 20 min. Cells were then lysed and postnuclear supernatants weresubjected to SDS-PAGE and Western blotting. Closed symbols denoteanti-CD3ε mAb-induced tyrosine phosphorylated proteins.

[0072]FIG. 11 is a Western blot using anti-phosphotyrosine mAb whichdemonstrates that CCS inhibits tyrosine phosphorylation inTCR-stimulated T cells. Th0 cells were treated with CCS (0 to 5 μg/ml)for 30 min, washed and then crosslinked anti-CD3ε mAb for 5 min. Cellswere then lysed and postnuclear supernatants were subjected to SDS-PAGEand Western blotting. In this figure, the symbols denote anti-CD3εmAb-induced tyrosine phosphorylation of ERK-2, which is reduced by CCS.

[0073]FIG. 12 is a Western blot using anti-Raf-1 mAb which shows thatCCS inhibits the mobility shift of Raf-1. . Th0 cells were treated withCCS (0 to 50 μg/ml) for 30 min, washed and then stimulated with either(A) combined PMA plus ionophore or (B) then crosslinked anti-CD3ε mAbfor 5 min. Cells were then lysed and postnuclear supernatants weresubjected to SDS-PAGE and Western blotting.

[0074]FIG. 13 is a pair of plots showing that CCS decreases IL-2production and proliferation in purified CD4⁺ T cells. T cells weretreated with CCS (50 μg/ml), washed and then cultured in either mediaalone or with immobilised anti-CD3ε mAb and soluble anti-CD28 mAb. (A)IL-2 production was determined by the CTL-L assay as described inExample 5. (B) Proliferation was determined by the incorporation of³H-thymidine. CD4+T cells cultured in the absence of mAb (stimuli) didnot produce any detectable IL-2 and do no proliferate.

[0075]FIG. 14 is a pair of plots showing that CCS decreases IL-2production by splenocytes but does not inhibit splenocyte proliferation.Splenocytes were treated with CCS (50 μg/ml), washed and then culturedin either media alone or with immobilised anti-CD3ε mAb. (A) IL-2production was determined by the CTL-L assay as described in Example 5.(B) Proliferation was determined by the incorporation of ³H-thymidine.Splenocytes cultured in the absence of mAb (stimuli) did not produce anydetectable IL-2 and do no proliferate).

[0076]FIG. 15 is a plot which shows that CCS inhibits tumour cell growthin vitro. Cancer cell lines were treated with CCS (50, 10, 2.5, 1 and0.25 μg/ml) or water as a control. After 96 h treatment, the effect ofCCS on tumour cell growth was evaluated. Columns represent the 50%inhibitory concentrations (IC₅₀ μg/ml) of CCS (the amount of CCSrequired to inhibit 50% of tumour cell growth.

EXAMPLE 1—Purification of Bromelain Proteins

[0077] a. Materials

[0078] Reagents Bromelain (E.C 3.4.22.4; proteolytic activity, 1,541nmol/min/mg) was obtained from Solvay Inc. (Germany). Fast Flow SSepharose, Pharmalyte 3-10™, Ampholine 9-11™, Ready Mix IEF™(acrylamide, bisacrylamide) and IEF™ markers were obtained fromPharmacia Biotech. Precast 4-20% acrylamide gels and broad rangemolecular weight markers were obtained from Bio-Rad Laboratories. Allother reagents were of analytical grade and obtained from either SigmaChemical Co. or British Drug House.

[0079] b. Proteinase Assay

[0080] The proteolytic activity of bromelain was determined by use of anin-house microtitre plate based assay using the synthetic substrateZ-Arg-Arg-pNA. This assay was based on that described by Filippova et alin Anal. Biochem., 143, 293-297 (1984). The substrate was Z-Arg-Arg-pNAas described by Napper et al in Biochem. J., 301, 727-735, (1994).

[0081] c. Protein Assay

[0082] Protein was measured using a kit supplied by Bio-Rad that is amodified method of Lowry et al (J. Biol. Chem. (1951) 193, 265-275).Samples were compared to bovine serum albumin standards (0 to 1.5 mg/ml)prepared in either 0.9% saline or 20 mM acetate buffer pH 5.0, asappropriate.

[0083] d. Preparation of Bromelain

[0084] All the following steps were performed at ambient temperature (20to 25° C.). A solution of bromelain (30 mg/ml) was prepared bydissolving 450 mg of powder in 15 ml of 20 mM acetate buffer (pH 5.0)containing 0.1 mM EDTA, sodium. The solution was dispensed into 10×1.5ml microcentrifuge tubes and centrifuged at 13,000×g for 10 minutes toremove insoluble material. The clear supernatants were pooled and usedfor chromatography

[0085] e. Fast Flow S-Sepharose High Performance Chromatography

[0086] A Fast flow S-sepharose column was prepared by packing 25 ml ofmedia into an XK 16/20™ column (Pharmacia Biotech) and equilibrated with20 mM acetate buffer (pH 5.0) containing 0.1 mM EDTA on an FPLC™ systemat 3ml/min. 5 ml of bromelain solution was injected onto the column.Unbound protein was collected and the column washed with 100 ml ofacetate buffer. Protein bound to the column was eluted with a lineargradient of 0 to 0.8 M NaCl in acetate buffer over 300 ml. 5 mlfractions were collected throughout the gradient and FIG. 2 shows atypical U.V. chromatogram of crude bromelain obtained from thisprocedure.

[0087] The fractions were then analysed for protein and proteolyticactivity as described above and FIG. 3 shows the proteolytic activityagainst the synthetic peptide Z-Arg-Arg-pNA and the protein content ofthe individual fractions. The protein content profile closely mirrorsthat of the U.V., as expected, but the main proteolytic activity isconfined to the two major peaks that correspond to that of bromelainprotease (SBP). Small activities are observed in other areas of thechromatogram that may corresponds to other proteases distinct from SBP,such as the later eluting CCS fraction, which contains ananain andcomosain.

[0088] The main peaks identified from the U.V. profile were pooled fromthree successive runs and named as indicated in Table 1. Pooledfractions were used for physico-chemical characterisation. Pooledfractions were concentrated by ultrafiltration and buffer exchangedusing PD10 columns into isotonic saline (0.9% w/v NaCl). The proteincontent and Z-Arg-Arg-pNA activity were calculated prior to biologicaltesting and are shown in Table 2.

[0089] The pooled fractions were processed for analysis as describedbelow. TABLE 1 Summary of Pooled Fractions from SP Sepharose HPFractionated Bromelain (QC2322) Fractions Pooled Component Description(Inclusive) CCT Flow through (unbound Unbound column components) flowthrough CCV First peak off column 8-9 CCX Second sharp peak off column13-14 CCZ Small peak on ascending edge of the 19-20 third main bromelainpeak CCY First main bromelain peak 23-24 CCW Second main bromelain peak27-29 CCU Small peak on descending edge of 33-34 the second mainbromelain peak CCS Last double peak off column 39-44

[0090] TABLE 2 Calculated Protein Content and Z-Arg-Arg-pNA Activity ofPooled Fractions used for Testing Biological Activity. PooledZ-Arg-Arg-pNA Activity Protein Content Fractions (μMoles/min/ml) (mg/ml)CCT 11.30 1.00 CCV 9.78 1.00 CCX 71.71 1.00 CCZ 688.81 1.00 CCY 1500.00.574 CCW 1500.0 0.543 CCU 1500.0 0.421 CCS 379.76 1.00

[0091] f. Processing of Pooled Fractions

[0092] The proteolytic activity and protein content of pooled fractionswere determined and the concentrations adjusted to approximately either1.4 mg/ml of protein or 105 nmoles/min/ml of proteinase activity using aFiltron™ stirred cell containing an ultrafiltration membrane of nominalmolecular weight cut-off of 10 kDa. The fractions were then bufferexchanged using PD10™ columns (Pharmacia Biotech) into isotonic saline(0.9% w/v NaCl), sterile filtered (0.2 μm) and adjusted for proteincontent or proteolytic activity. Samples were then frozen at −80° C. andused in the in vitro studies described below.

[0093] g. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis(SDS-PAGE)

[0094] Pooled FPLC™ samples were analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) on precast 4 to 20% Tgradient gels. Samples were prepared for electrophoresis by acidprecipitation in which 100 μl was mixed with an equal volume of 20% w/vtrichloroacetic acid (TCA). Precipitated protein was collected bycentrifugation at 13,000×g for 10 minutes and the supernatant discarded.The pellet was washed twice with 0.5 ml of diethyl ether and left to dryin air at ambient temperature. The pellets were then dissolved in 300 μlof SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8 containing 10% v/vglycerol, 2% w/v sodium dodecyl sulphate and 40 mM dithiothreitol) andheated at 95° C. in a water bath.

[0095] SDS-PAGE broad range molecular weight standards diluted 1:20 inSDS-PAGE sample buffer were treated similarly and run with the samples.Gels were run on a mini Protean II™ electrophoresis system according toBio-Rad's protocol at 240 V and until the dye front reached the end ofthe gel (30 to 45 min).

[0096] After electrophoresis, separated proteins were stained overnightwith orbital mixing in a solution of 0.075% w/v colloidal brilliant blueG-250 containing 1.5% v/v phosphoric acid, 11.25% w/v ammonium sulphateand 25% v/v methanol. Gels were destained, to obtain a clear background,in a solution of 25% v/v methanol and 10% v/v acetic acid.

RESULTS

[0097] The purity of fractions is shown by SDS-PAGE in FIG. 4. All ofthe pooled fractions except the column flow through (CCT) showed thatthe major protein present was of molecular weight between approximately25-28 kDa. This corresponds to the molecular weight of cysteineproteinases isolated from bromelain by other authors (Rowan et al,Methods in Enzymology, (1994), 244, 555-568). The purity of fractionsCCX, CCZ, CCY and CCW appears to be high. Minor components of lowermolecular weight can be observed in some fractions, particularly CCT,CCV, CCX and CCS. Pooled fractions CCU and CCS contain a doublet between25-28 kDa; the higher gel loadings of fractions CCX, CCZ, CCY, and CCWmeans that doublet bands may also be present in these fractions. Asummary of the components and their calculated molecular weights inpooled fractions, as determined by SDS-PAGE, is shown in Table 3.

[0098] Proteins in pooled fractions CCX, CCZ, CCY+CCW and CCU weretransferred onto nitro-cellulose after SDS-PAGE by Western blotting andprobed with rabbit antisera raised against purified stem bromelainprotease (SBP) (results not shown). All protein bands in these pooledfractions were recognised by antibodies in the sera, indicatingimmunologically similar proteins, probably belonging to the cysteineproteinase family of enzymes. TABLE 3 Summary of the Molecular Weightsof Proteins found in SP Sepharose HP Pooled Fractions as Determined bySDS-PAGE. Pooled Molecular Weight (kDa) of Molecular Weight (kDa) ofFractions Major Protein Band(s) Minor Protein Band(s) CCT 76.03 15.07CCV 15.07, 25.85, 28.28, 76.03 CCX 25.08 15.07, 76.03 CCZ 27.45 13.37,16.49, 76.03 CCY 27.45 6.5 CCW 27.45 CCU 27.45, 28.28 CCS 15.07, 25.85,27.45

[0099] h. Isoelectric Focusing

[0100] Pooled fractions (0.5 to 1.0 mg/ml) were diluted 1:3 withdeionised water and run on gradient gels of pH 3 to 11. Gels were castusing Ready Mix IEF™ to produce a 5.5% T, 3% C polyacrylamide gelcontaining 10% v/v glycerol, 5.0% Pharmalyte 3-10™ and 2.5% Ampholine9-11™. Briefly, 10 μ; of sample and high pI markers were loaded onto thegel after prefocusing at 700 V. Sample entry was at 500 V for 10 min,focusing was at 2500 V for 1.5 hour and band sharpening at 3000 V for 10min. After electrophoresis the proteins were fixed with a solution of20% w/v TCA for 30 min, washed in destain for 30 min to remove TCA andstained with brilliant blue G-250 as described for SDS-PAGE (see above).

RESULTS

[0101]FIG. 5 shows that all fractions except CCX contained basicproteins focusing beyond the 9.3 pI marker. Localised chargeinteractions with the chromatographic media functional groups mayexplain why proteins of pI 3.8 and 3.85 in CCX, adsorbed onto a cationexchange resin at pH 5.0. CCZ was present as a single band of pI 9.7,whilst pooled fractions CCY, CCW, and CCU contained multiple bands ofisoelectric points in the range pH 9.5-9.8. At least part of thisheterogeneity can be explained by variation in the carbohydrate moietyof a common stem bromelain protein backbone. The values are in agreementwith those reported in the literature of pI 9.45-9.55 for bromelain(Rowan et al, Methods in Enzymology, (1994), 244, 555-568). Pooledfractions CCS contains two basic proteins of pI greater than 10.25.Estimates by extrapolation give pIs of 10.4 and 10.45. These correspondto ananain and comosain, and are in agreement with other estimates(Rowan et al, as above) of pIs greater than 10. The pIs of proteins ineach of the pooled fractions are summarised in Table 4. TABLE 4 Summaryof the estimated Isoelectric points of Proteins found in SP Sepharose HPPooled Fractions. Pooled Fractions Isoelectric Points of Proteins CCTNot detected CCV Not Detected CCX 3.8, 3.85 CCZ 9.7 CCY 9.6, 9.7 CCW9.57, 9.6, 9.7 CCU 9.57, 9.6, 9.75 CCS 10.4, 10.45

EXAMPLE 2—NH₂-terminal Amino Acid Analysis of Bromelain Components

[0102] In a separate experiment, pooled fractions of bromelain were runby SDS PAGE and blotted as above onto PVDF membrane. The membrane wasstained with 0.025% w/v coomassie blue R-250, dissolved in 40% v/vmethanol for 10 min, followed by destaining in 50% v/v methanol. Themembrane was dried in air at room temperature and NH₂-terminal aminoacid sequencing of the stained proteins was carried out. Briefly, theprotein band was cut from the membrane and placed in the upper cartridgeof the sequencer. NH₂-terminal amino acid analysis of bromelaincomponents was determined by Edman degradation using a gas phasesequencer (Applied Biosystems), equipped with an on-linephenylthiohydantion amino acid analyser. Table 5 shows the first 21NH₂-terminal amino acids of CCZ, CCZ, stem bromelain protease, ananainand comosain. TABLE 5 NH₂-Terminal Sequence Similarities of CCZ Proteinand Those of Known Proteinases Isolated from Bromelain. ProteinasePosition from N-Terminus CCZ VLPDSIDWRQKGAVTEVKNRG 1  5  10  15  20 CCXVPQSIDWRDYGAVNEVKN   4  9  14 Stem Bromelain AVPQSIDWRDYGAVTSVKNQNProtease 1  5  10  15  20 Ananain VPQSIDWRDSGAVTSVKNQG 1  4  9  14  19Comosain VPQSIDWRNYGAVTSVKNQG 1  4  9  14  19

[0103] All proteins share sequence homologies. Ananain and comosaindiffer by 2 out of 20 amino acids when compared to stem bromelainprotease. CCZ differs by 8 out of 21 amino acids when compared to stembromelain protease. CCZ differs from ananain and comosain by 6 out of 20amino acids. Comosain differs by 2 amino acids from ananain. Whilst itis clear that these proteins are structurally related, they are alldistinct, showing divergence from each other. These proteinases alsodiffer in their proteinase substrate specificity and their biologicalactivity.

EXAMPLE 3—Fraction CCS inhibits tyrosine phosphorylation of p42 kdaphosphoprotein

[0104] a. Materials

[0105] Antibodies Anti-CD3 ε-chain mAb (145-2C11) and anti-CD28 mAb(PV-1) were purchased from Pharmingen (San Diego, Calif.) and goatanti-hamster IgG Ab was from Sigma (Dorset, UK). Mouseanti-phosphotyrosine mAb (4G10), mouse anti-MAPk R2 (ERK-2) mAb andmouse anti-Raf-1 mAb were from UBI (Lake Placid, N.Y.). Goat anti-mouseand goat anti-rabbit IgG Ab conjugated to horse radish peroxidase (HRP)were from BioRad (Hemel Hemstead, Hertfordshire, UK). Rabbit polyclonalphospho-specific MAPk IgG which recognise tyrosine phosphorylated p44and p42 MAPks were from New England BioLabs (Hitchin, Hertfordshire,UK).

[0106] Reagents Phorbol 12-myristate 13-acetate (PMA) and calciumionophore A23187 were purchased from Sigma. Bromelain (E.C 3.4.22.4;proteolytic activity, 1,541 nmol/min/mg) was obtained from Solvay Inc.(Germany). E-64 (L-trans-epoxysuccinylleucylamido-(4-guanidino)butane, aselective cysteine protease inhibitor, was from Sigma.

[0107] Cells The T cell hybridoma GA15 was a generous gift from B. Fox(ImmuLogic Pharmaceutical Corporation, Boston, Mass.). GA15 wasgenerated by fusing the thymoma BW5147 with the T_(h)2 clone F4 specificfor KLH in association with I-Ab, and were maintained as previouslydescribed (Fox, 1993, Int. Immunol., 5, 323-330). GA15 exhibit a Th0cell phenotype as they produce IL-2, IL-4 and IFN-γ followingstimulation with crosslinked anti-CD3ε mAb (Fox, 1993).

[0108] b. Stimulation of T cells. Cells (2×10⁷) suspended in RPMI 1640were treated with CCS (1 to 50 μg/ml) diluted in saline (0.9% (w/v)) for30 min at 37° C. Mock treated cells were treated with an equal volume ofsaline (diluent). At high concentrations of CCS (50 or 100 μg/ml) cellaggregation occurred, as noted previously in studies with crudebromelain. Following treatment, cell aggregates were gently dispersed bywashing cells 3 times and then resuspending in fresh RPMI. Cells werestimulated via the cell surface with crosslinked mAb to the TCR(anti-CD3ε), or directly, using combined PMA (20 ng/ml) and ionophore (1μM) for times indicated in figure legends and the text.

[0109] Stimulation via the TCR was conducted by first incubating T cellson ice for 30 min with anti-CD3ε mAb (20 pig/ml). Excess mAb was thenremoved by washing once at 4° C. and anti-CD3s mAb was crosslinked withgoat anti-hamster IgG (20 μg/ml) at 37° C. Stimulation was terminated bythe addition of ice-cold lysis buffer (25 mM Tris, pH 7.4, 75 mM NaCl, 2mM EDTA, 0.5% Triton X-100, 2 mM sodium orthovanadate, 10 mM sodiumfluoride, 10 mM sodium pyrophosphate, 74 μg/ml leupeptin, 740 μM PMSFand 74 μg/ml aprotinin) for 30 min with continual rotation at 4° C.Lysates were clarified (14,000×g for 10 min) and an equal volume of 2×SDS-PAGE sample buffer (50 mM Tris, pH 7, 700 mM 2-ME, 50% (v/v)glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) was added topostnuclear supernatants. Proteins were solubilised at 100° C. for 5 minand samples containing 1×10⁶ cell equivalents were resolved by SDS-PAGE.

[0110] c. Immunoblotting. Separated proteins were transferred tonitrocellulose membranes (Bio-Rad) which were then blocked with 5% (w/v)bovine serum albumin (Sigma, fraction V; BSA), 0.1% Nonidet p40™ inTris-buffered saline (170 mM NaCl and 50 mM Tris, pH 7.4; TBS).Immunoblots were incubated with the appropriate antibodies as indicatedin figure legends. Primary antibodies were diluted in antibody dilutionbuffer comprised of 0.5% (w/v) BSA, 0.1% (v/v) Tween-20 in TBS at 4° C.for 2 h followed by detection with the appropriate secondary antibodyconjugated to horseradish peroxidase diluted in antibody dilution bufferat 4° C. for 1 h. Following each incubation step, membranes were washedextensively with 0.1% Tween-20 in TBS. Immunoreactivity was determinedusing the ECL chemiluminescence detection system (Amersham Corp.,Arlington Heights, Ill.).

[0111] d. Inhibition of proteolytic activity of CCS. A specific cysteineprotease inhibitor, E-64, was used to inactivate the proteolyticactivity of CCS. CCS (25 μg/ml) diluted in 3 μM dithiothreitol, 100 μME-64, 60 mM sodium acetate (pH 5) was incubated for 10 minutes at 30° C.The inactivated CCS was then dialysed overnight in saline at 4° C.Earlier studies with crude bromelain have shown that these conditionsare sufficient to induce 99.5% inactivation of proteolytic activity asassayed with the Z-Arg-Arg-pNA substrate (see above). T cells weretreated with E-64 inactivated CCS (25 μg/ml) and compared with untreatedCCS and mock-treated T cells stimulated with PMA plus ionophore.

RESULTS

[0112] a. Fraction CCS inhibits tyrosine phosphorylation of p42 kdaphosphoprotein. We have previously shown that bromelain blocks tyrosinephosphorylation of ERK-2 following stimulation of T cells with combinedPMA plus calcium ionophore (WO-A-96/00082). Phorbol ester and ionophorestimulation of T cells act synergistically to reproduce many features ofTCR stimulation such as IL-2 secretion, IL-2 receptor expression, and Tcell proliferation (Truneh et al., 1985, Nature, 313, 318-320; Rayter etal., 1992, EMBO, 11, 4549-4556). Phorbol esters can mimic antigenreceptor triggering and bypass TCR-induced protein tyrosine kinases toactivate ERK-2 by a direct agonist action on PKC and p21^(Ras). Calciumionophore A23187 induces increased intracellular release of Ca²⁺ andtherefore mimics the action of inositol 1,4,5-trisphosphate (IP₃).Phorbol esters and ionophore however, stimulate PKC pathways that arenot controlled by the TCR (Izquierdo et al., 1992, Mol. Cell. Biol., 12,3305-3312) suggesting separate intracellular pathways within T cellsthat regulate T cell function. We therefore investigated which fractionof bromelain could block T cell signalling via the TCR-independentpathway by examining its effect on PMA and ionophore-induced tyrosinephosphorylation.

[0113] Stimulation of T cells with combined ionophore and PMA inducedtyrosine phosphorylation of several proteins including those of circa100 kda, 85 kda, 42 kda and 38 kda. CCS (50 μg/ml) pre-treatment reducedtyrosine phosphorylation of the p42 kda protein, and did notsignificantly inhibit phosphorylation of any other substrate (FIG. 6).In two experiments, CCS, but no other fraction, also increased tyrosinephosphorylation of circa 36 kda, 38 kda, 85 kda, 94 kda and 102 kdaproteins (FIG. 7 and FIG. 8).

[0114] The ability of CCS to block tyrosine phosphorylation of the 42kda phosphoprotein was dose-dependent (FIG. 8) and dependent on itsproteolytic activity, since E-64 completely abrogated the inhibitoryeffect of CCS on p42 kda phosphorylation (FIG. 8). E-64 treatment of Tcells did not affect PMA and ionophore-induced T cell signalling.

[0115] CCS inhibits ERK-2 tyrosine phosphorylation. We suspected thatthe 42 kda phosphoprotein inhibited by CCS was the MAPk ERK-2. so weconducted immunoblot analysis with specific anti-ERK-2 mAb andanti-phospho MAPk antibodies, which specifically detects ERK-1 and ERK-2only when catalytically activated by phosphorylation at Tyr204.Immunoblotting of CCS-treated cells that had been stimulated with PMAplus ionophore, confirmed that the p42 kda phosphoprotein was indeedERK-2 (FIG. 9).

[0116] CCS reduces TCR-induced tyrosine phosphorylation of ERK. We nextinvestigated the effect of CCS on TCR-mediated signal transduction byassessing substrate tyrosine phosphorylation of GA15 stimulated withcrosslinked anti-CD3E mAb. Immunoblots of GA15 lysates, using specificanti-phosphotyrosine mAb, revealed increased tyrosine phosphorylation ofmultiple proteins including those of circa 120 kda, 100 kda, 85 kda, 76kda, 70 kda, 42 kda and 40 kda, consistent with phosphoproteins observedin other T cell lines following TCR-ligation (June et al, 1990, J.Immunol., 144, 1591-1599 and Proc. Natl. Acad. Sci. USA, 87, 7722-7726,reviewed by Cantrell, 1996, Annu. Rev. Immunol., 14, 259-274) (FIG. 10).Tyrosine phosphorylated proteins were readily detected between 2 and 5min following stimulation and remained phosphorylated for at least 10min (FIG. 10). GA15 cells stimulated with anti-CD3 mAb alone orcross-linking Ab, did not induce tyrosine phosphorylation of anycellular substrate (data not shown). Again, CCS pre-treatment of GA15for 30 min caused a reduction in TCR-induced protein tyrosinephosphorylation of ERK-2 in a dose-dependent manner (FIG. 11). CCS didnot markedly affect tyrosine phosphorylation of other TCR-inducedphosphoproteins, suggesting that CCS has a selective mode of action.

EXAMPLE 4—CCS retards the mobility shift of Raf-1

[0117] Raf-1 is an immediate upstream activator of MEK-1 which activatesERK-2. Raf-1 activation requires phosphorylation on specific serine andthreonine residues (Avruch et al., 1994, TIBS, 19, 279-283). Toinvestigate whether CCS affects any other substrates upstream from ERK-2in the MAP kinase cascade, we investigated the effect of CCS on Raf-1. Tcells were treated with CCS (0 to 50 μg/ml) and then stimulated witheither anti-CD3ε mAb or combined PMA plus ionophore as describedearlier. Results show that CCS blocks the mobility shift of Raf-1,indicating that it blocks its protein phosphorylation and thusactivation. This data confirms that CCS has an effect on the MAP kinasecascade (FIG. 12) and that the effect of CCS may not be directly onERK-2, but on upstream substrates in the MAPk cascade.

EXAMPLE 5—Effect of CCS on IL-2 Production and T cell Proliferation

[0118] a. Materials

[0119] Cells Splenocytes were isolated from female BALB/c mice (6-8weeks old), as previously described in WO-A-96/00082. Highly purifiedCD4⁺ T cells were isolated from splenocytes using magnetic activatedcell sorting (MACS).

[0120] b. Interleukin 2 production. T cells diluted in RPMI were treatedwith CCS (50 μg/ml) or saline at 37° C. for 30 min, washed in fresh RPMIand then resuspended in culture medium. T cells were stimulated toproduce cytokine mRNA by immobilised anti-CD3s (4 μg/ml) and solubleanti-CD28 (10 μg/ml). Anti-CD3ε mAb diluted in PBS was immobilised to24-well, flat bottom, microculture plates (Corning, Corning, N.Y.) byincubation for 16 hours at 4° C. Wells were then washed three times inPBS prior to addition of triplicate cultures of either splenocytes orpurified CD4+T cells (2.5-5×10⁶ cells per well) which were incubated at37° C. in humidified 5% CO₂ for 24 h. IL-2 levels in the culturesupernatant were measured using the CTL-L bioassay (Gillis et al., 1978,J. Immunol., 120, 2027-2032).

[0121] c. T Cell Proliferation. T cells were treated with CCS (50 μg/ml)for 30 min, washed in RPMI then stimulated with immobilised anti-CD3εmAb alone or combined anti-CD3ε mAb plus anti-CD28 mAb. Cells were thencultured in 96 well, flat-bottom plates (Nunc) at 10⁵ cells per well for36 h. Cultures were pulsed with 0.5 μCi of [³H]TdR 12 h prior toharvesting onto glass fibre filters.

RESULTS

[0122] a. CCS inhibits IL-2 production and proliferation of CD4⁺ Tcells.

[0123] Activation of p21^(Ras), Raf-1, MEK-1 and ERKs are essential forinduction of IL-2 transcription in T cells (Izquierdo et al., 1993, J.Exp. Med., 178, 1199). IL-2 is the major autocrine T cell growth factorwhich induces proliferation of T cells. The defect in ERK activationdemonstrated here could therefore be expected to inhibit IL-2 productionand T cell proliferation. We therefore investigated whether CCS couldeffect a functional outcome of T cell signalling, namely IL-2 productionand proliferation in murine splenocytes and highly purified CD4⁺ Tcells. CCS (50 μg/ml) treatment of purified CD4+T cells reduced bothIL-2 production and proliferation when the ERK pathway was stimulatedwith anti-CD3ε mAb (FIG. 13a and 13 b). CCS also blocked IL-2 productionby splenocytes, however it did not affect splenocyte proliferation (FIG.14a and 14 b), suggesting that an as yet unidentified component in CCSwas acting on accessory cell populations in splenocyte cultures, such asB cells or macrophages. Bromelain can increase costimulatory signals toT cells via an action on B cells. Regardless of the putative effect ofCCS on accessory cells, data clearly indicate that CCS blocks IL-2production and proliferation of purified CD4⁺ T cells, suggesting thatCCS blocks T cell activation. IL-2 production and proliferation weredependent on cell stimulation with anti-TCR antibodies as no cytokinewas detected in cells cultured in tissue culture media alone (FIG. 13and 14).

EXAMPLE 6—Effect of CCS on Human Tumour Cell Growth in vitro

[0124] a. Materials

[0125] Cells Tumour cell lines were provided by L. Kelland (Institute ofCancer Research, Sutton, UK) and were as follows; ovarian (SKOV-3, CH-1,A2780), colon (HT29, BE, LOVO), breast (MCF-7, MDA231, MDA361), lung(A549, CORL23, MOR) and melanoma (G361, BOO8, SKMe124).

[0126] b. Growth Inhibition of Human Tumour Cell Lines. Studies wereconducted by L. Kelland (Institute of Cancer Research, Sutton, UK). Celllines were trypsinised and single viable cells were seeded into 96-wellmicrotitre plates at a density of 4×10³ cells/well in 160 μl growthmedium. After allowing for attachment overnight, CCS was then added toquadruplicate wells in 40 μl of growth medium to give a range of finalconcentrations in wells of 50, 10, 2.5, 1 and 0.25 μg/ml. Eight wellsserved as control, untreated wells. CCS was diluted immediately prior toaddition to cells in sterile water. CCS exposure to cells was for 96 hwhereupon the cell number in each well was determined by staining with0.4% sulforhodamine B in 1% acetic acid as described previously (Kellandet al., 1993, Cancer Res., 53, 2581-2586). 50% inhibitory concentrations(IC₅₀ values in μg/ml) were then calculated from plots of concentrationversus control (%) absorbance (read at 540_(nm)).

RESULTS

[0127] a. CCS inhibits human tumour growth in vitro. p21Ras and Raf-1are important oncogenes, which when mutated cause uncontrolled cellgrowth and proliferation, leading to cancer. Since we have shown thatCCS can block the effects of the p21Ras/Raf-1/MEK1/ERK kinase signallingcascade, we investigated whether CCS could block tumour growth. CCStreatment of human tumour cells resulted in a reduction in the growth ofseveral different ovarian, lung, colon, breast and melanoma tumour celllines in vitro (FIG. 15). CCS did not affect all cell lines equally,suggesting that CCS has a selective action.

1 5 1 21 PRT ORGANISM Ananas comosus 1 Val Leu Pro Asp Ser Ile Asp TrpArg Gln Lys Gly Ala Val Thr Glu 1 5 10 15 Val Lys Asn Arg Gly 20 2 18PRT ORGANISM Ananas comosus 2 Val Pro Gln Ser Ile Asp Trp Arg Asp TyrGly Ala Val Asn Glu 1 5 10 15 Val Lys Asn 3 21 PRT ORGANISM Ananascomosus 3 Ala Val Pro Gln Ser Ile Asp Trp Arg Asp Tyr Gly Ala Val ThrSer 1 5 10 15 Val Lys Asn Gln Asn 20 4 20 PRT ORGANISM Ananas comosus 4Val Pro Gln Ser Ile Asp Trp Arg Asp Ser Gly Ala Val Thr Ser Val 1 5 1015 Lys Asn Gln Gly 20 5 20 PRT ORGANISM Ananas comosus 5 Val Pro Gln SerIle Asp Trp Arg Asn Tyr Gly Ala Val Thr Ser Val 1 5 10 15 Lys Asn GlnGly 20

1. A component of bromelain which contains proteins having molecularweights of about 15.07 kDa, 25.85 kDa and 27.45 kDa as determined bySDS-PAGE, has isoelectric points of 10.4 and 10.45 and is obtainable bythe following method: i. dissolving bromelain in acetate buffer at pH5.0; ii. separating the components of the bromelain by fast flow highperformance liquid chromatography on S-sepharose eluting with a lineargradient of 0 to 0.8 M sodium chloride in acetate buffer over 300 ml;iii. collecting the fraction corresponding to the final double peak offthe column; and iv. isolating the protein from the fraction collected in(iii).
 2. A component of bromelain as claimed in claim 1, for use inmedicine, particularly in the treatment or prevention of diseases andconditions mediated by: i. activation of T cells; ii. activation of theMAP kinase pathway; or iii. the production of growth factors orcytokines; or in the treatment or prevention of cancer.
 3. A componentof bromelain as claimed in claim 1 for modulating pathways controllingcell growth and proliferation.
 4. A component of bromelain as claimed inclaim 1 for inhibiting the production of growth factors or cytokines bycells.
 5. A component of bromelain as claimed in claim 1 for inhibitingthe activation of the MAP kinase pathway.
 6. A component of bromelain asclaimed in claim 1 for inhibiting the activation of T cells.
 7. Acomponent of bromelain as claimed in claim 1 for use as animmunosuppressant.
 8. A component of bromelain as claimed in claim 1 forblocking the production of growth factors and cytokines or for thetreatment or prevention of autoimmune diseases, graft or transplantrejection by a host, allergic reactions, toxic shock, apoptosis,parasite or pathogen infections or cancer.
 9. The use of ananain,comosain, a mixture of ananain and comosain or a component of bromelainas claimed in claim 1 in the preparation of an agent for modulatingintracellular signalling pathways which control cell growth andproliferation.
 10. The use of ananain, comosain, a mixture of ananainand comosain or a component of bromelain as claimed in claim 1 in thepreparation of an agent for inhibiting the production of growth factorsand cytokines by cells.
 11. The use of ananain, comosain, a mixture ofananain and comosain or a component of bromelain as claimed in claim 1in the preparation of an agent for reducing or preventing the activationof the MAP kinase pathway.
 12. The use of ananain, comosain, a mixtureof ananain and comosain or a component of bromelain as claimed in claim1in the preparation of an agent for reducing or preventing theactivation of T cells.
 13. The use of ananain, comosain, a mixture ofananain and comosain or a component of bromelain as claimed in claim 1,wherein the agent is an immunosuppressive agent.
 14. The use of ananain,comosain, a mixture of ananain and comosain or the CCS fraction ofbromelain in the preparation of an agent for the treatment or preventionof diseases and conditions mediated by i. the activation of T cells; ii.activation of the MAP kinase pathway; or ii. the production of growthfactors or cytokines; or in the treatment or prevention of cancer. 15.The use of ananain, comosain, a mixture of ananain and comosain or acomponent of bromelain as claimed in claim 1 in the preparation of anagent for blocking the production of growth factors and other cytokinesor for the treatment or prevention of autoimmune diseases, graft ortransplant rejection by a host, allergic reactions, toxic shock,apoptosis, parasite or pathogen infections or cancer.
 16. Apharmaceutical or veterinary composition comprising the CCS fraction ofbromelain together with a pharmaceutically or veterinarily acceptableexcipient.
 17. A pharmaceutical or veterinary composition as claimed inclaim 16, which is adapted for enteral, for example oral, nasal, buccal,topical or anal administration.
 18. A pharmaceutical or veterinarycomposition as claimed in claim 16, which is adapted for parenteraladministration, for example by the intravenous, subcutaneous,intramuscular or intraperitoneal routes.
 19. A pharmaceutical orveterinary composition as claimed in claim 16 which is adapted for oraladministration and which is an enteric coated preparation.